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研究生:林泰都
研究生(外文):Tai-Tu Lin
論文名稱:表皮生長因子受器在高糖及第ㄧ型乙型生長因子誘導NRK-49F細胞生長與膠原蛋白產生之角色
論文名稱(外文):Role of EGFR in high glucose and TGF-β1-induced cell growth and collagen production in NRK-49F cells
指導教授:莊麗月莊麗月引用關係
指導教授(外文):Lea-Yea Chuang
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:生物化學研究所碩士班
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:107
中文關鍵詞:表皮生長因子受器第ㄧ型乙型生長因子
外文關鍵詞:EGFRTGF-β1NRK-49F cells
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糖尿病腎病變 (diabetic nephropathy) 是最常見的糖尿病併發症之一,目前已知造成糖尿病腎病變的因子有:高血糖、高度糖化終產物 (advanced glycation end-product, AGE)、第二型血管張力素 (angiotensin II, Ang II) 與第一型乙型轉型生長因子 (transforming growth factor-β1, TGF-β1) 等, 而高血糖、AGE、Ang II、和氧化壓力等,皆會影響 TGF-β1 的表現。本實驗室首先發現上皮生長因子 (epidermal growth factor, EGF) 與上皮生長因子接受器(epidermal growth factor receptor, EGFR) 在糖尿病腎病變有過度表現的情形,因此本篇研究即探討 EGFR 在糖尿病腎病變所扮演的角色。
利用 Western blot 與 real-time PCR 實驗發現, TGF-β1 刺激正常大鼠腎臟纖維母細胞 (NRK-49F) 會造成 EGFR 的磷酸化以及 EGFR mRNA 和蛋白質的合成;且促進細胞 collagen 合成並誘發細胞增生。當我們加入 EGFR 專一性的抑制劑 Iressa 時, Iressa 不僅抑制 EGFR 的活化,也抑制了由 TGF-β1 所引起的 collagen 合成與細胞增生的效應。進一步我們發現 TGF-β1 可以經由活化 EGFR 而誘導 FGF2 與 PAI-1 的表現。利用不同訊息傳遞的抑制劑,如:TGF-β type I receptor inhibitor (SB431542)、p38 inhibitor (SB203580)、EKR1/2 inhibitor (PD98059) 等,發現 TGF-β type I receptor inhibitor 與 Iressa 可以抑制 FGF2 表現,並逆轉細胞 collagen 合成的效應。此外 Smad2、Smad3 dominant negative plasmid transfection 的技術,發現 TGF-β1 可經由 Smad2/3 轉錄因子活化 EGFR 再誘導 FGF-2 與 PAI-1 表現,進而刺激細胞外間質 (collagen type I、IV 與 fibronectin) 的合成並抑制細胞外間質分解而導致纖維化。另外對於 TGF-β1 所誘導 NRK-49F 細胞增生的效應,我們發現,TGF-β1 可經由活化 EGFR-ERK1/2 與 p38 訊息傳遞路徑而造成細胞增生。此外我們由 real-time PCR 與 luciferase assay 發現以高糖或 Ang II 刺激 NRK-49F 細胞,會誘使 TGF-β1 的表現與活性增加,由 3H-proline incorporation 與 3H-thymidine incorporation 也發現高糖或 Ang II 可能是經由誘導 TGF-β1 分泌、進而活化 EGFR 路徑而導致細胞 collagen 合成與增生的效應。另外,我們也首先評估 Iressa 對於延緩或治療糖尿病腎病變的可行性,實驗結果發現在 NRK-49F 細胞中,Iressa 可逆轉由高糖、Ang II 或 TGF-β1 所導致細胞collagen 合成與增生,且在小鼠腎間質細胞與正常大鼠腎小管上皮細胞也可以逆轉 TGF-β1 所導致細胞 collagen 合成的效應。
綜合本篇結果,我們首先提出 TGF-β1 須經由 Smad signaling 活化 EGFR 、增加 FGF2 分泌、刺激 PAI-1 大量表現,刺激細胞外間質合成與抑制細胞外間質分解而導致纖維化;且 TGF-β1 也可經由活化 EGFR 訊息路徑而使 ERK1/2 活化,造成細胞增生,而 Iressa 則可以做為延緩或治療糖尿病腎病變的潛力藥物。
Diabetic nephropathy (DN) is a common complication of diabetic mellitus. Hyperplycemia, advanced glycation end-product (AGE), angiotensin II (Ang II) and transforming growth factor beta1 (TGF-β1) play important roles in DN. TGF-β1 is induced by hyperplycemia, AGE, Ang II, and reactive oxygen species (ROS). Additionally, our laboratory have shown that both EGF (epidermal growth factor) and EGF receptor (EGFR) are overexpressed in DN rats. Thus, we wished to understand the role of EGFR in an in vitro model of DN.
We found that TGF-β1 phosphorylated EGFR and up-regulated EGFR mRNA and protein expression in NRK-49F cells. TGF-β1 also induced FGF-2 and PAI-1 expression via inducing p-EGFR. Inhibition of EGFR (Iressa) or Smad2/3 (by SB-431542 and Smad2、Smad3 dominant negative plasmid) attenuated TGF-β1-induced FGF-2 and PAI-1 expression. Thus, TGF-β1 induced FGF-2 and PAI-1 expression via EGFR and Smad2/3. Inhibiting EGFR also attenuated cell cycle change. Moreover, TGF-β1-inducd proliferation is accompanied by p38 and ERK phosphorylation. We found that TGF-β1 actived EGFR-ERK signaling to induce cell proliferation. Morevoer, hyperglycemia (HG) and Ang II induced TGF-β1 and collagen synthesis and induced cell proliferation.
Thus, TGF-β1 activated EGFR signaling through Smad2/3. Moreover, TGF-β1 induced FGF-2 and PAI-1 expression. FGF-2 induced extracellular matrix expression whereas PAI-1 decreases extracellular matrix degradation thereby inducing fibrosis. Iressa reversed TGF-β1-induced fibrosis and proliferation in NRK-49F cells. Thus, EGFR is involved in the pathogenesis of DN in this in vitro model.
目次
摘要………………………………………………………….2
縮寫表……………………………………………………….6
緒論………………………………………………………….9
研究目的…………………………………………………….22
材料與方法………………………………………………….25
實驗結果…………………………………………………….43
討論………………………………………………………….54
圖表………………………………………………………….64
參考文獻…………………………………………………….93
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